The invention relates to transgenic animals which express reporter genes coupled to promoters of genes involved in the health, aging, or appearance of the skin, e.g., theversican, VEGF, or MMP promoters, and methods of using such animals in evaluating treatments, e.g., compounds, for their effect on skin.
The inventors have discovered that transgenic animals having one or more constructs which include a skin-metabolism promoter coupled to a reporter gene can be used to evaluate a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for use in enhancing the health or appearance of the skin.
Accordingly, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for its effect on skin. The method includes:
providing a transgenic animal having a reporter gene coupled to a skin metabolism-related promoter, preferably a human promoter;
administering the treatment to the transgenic animal or a tissue therefrom; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments, the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or transgenic mammal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope. In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, see Takeda et al., 1991, L. Am. Geriatr. 39:911-919, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In preferred embodiments, the promoter is heterologous from the transgenic animal, i.e., the promoter is from another species. In other preferred embodiments the promoter is from the same species as the transgenic animal. In particularly preferred embodiments, the skin metabolism-related promoter is a human skin metabolism-related promoter. In particularly preferred embodiments, the skin metabolism-related promoter is: a promoter from a gene which encodes a transmembrane protein or a component of the extracellular matrix, such as a proteoglycan promoter, e.g., a versican promoter; a promoter from a protease expressed in the skin, e.g., a matrix metalloproteinase (MMP) promoter, e.g., an MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, or MMP9 promoter; a promoter from a gene which affects vascular function, e.g., a vascular endothelial growth factor promoter; a hyaluronan synthase promoter, e.g., a hyaluronan synthase 1 promoter, a hyaluronan synthase 2 promoter, or a hyaluronan synthase 3 promoter; a promoter for a collagenase expressed in the skin, e.g., a MMP2 or MMP9, preferably a MMP9, promoter; or a neutrophil elastase promoter.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments, the treatment is administered repeatedly, prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the transgenic animal further includes a second reporter gene coupled to a second skin metabolism-related promoter, wherein the second skin metabolism-related promoter is different from the first skin metabolism-related promoter. The reporter gene coupled to the first promoter can be the same or different from the reporter gene coupled to the second promoter. For example, the transgenic animal can include: a first reporter gene coupled to the versican promoter and a second reporter gene coupled to the vascular endothelial growth factor promoter; a first reporter gene coupled to the versican promoter and a second reporter gene coupled to a hyaluronan synthase promoter; a first reporter gene coupled to the hyaluronan synthase promoter and a second reporter gene coupled to the vascular endothelial growth factor promoter; a first reporter gene coupled to a matrix metalloproteinase (MMP) promoter and a second reporter gene coupled to a MMP2 or MMP9, preferably MMP9, promoter; a first reporter gene coupled to a matrix metalloproteinase (MMP) promoter and a second reporter gene coupled to the neutrophil elastase promoter; or a first reporter gene coupled to a MMP2 or MMP9, preferably a MMP9, promoter and a second reporter gene coupled to the neutrophil elastase promoter. In preferred embodiments, the transgenic animal can include two constructs both of which are upregulated. In preferred embodiments, the transgenic animal can include two constructs both of which are downregulated.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGFxcex2; of TGFxcex1.
In preferred embodiments, the method further includes administering a second treatment) to the transgenic animal. The second treatment can be one which injures or damages the skin, kills skin cells, or can include the removal of hair, e.g., by plucking, shaving, or application of a depilatory, or in general, induces an unwanted condition of the skin. The second treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the treatment can be determined in the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.
In another aspect, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound for its effect on skin. The method includes:
providing a transgenic animal, e.g., a mouse, having a reporter gene coupled to a, preferably human, versican promoter;
administering a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, to the transgenic animal, or to a tissue taken therefrom; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the versican promoter is a human versican promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments the treatment is administered repeatedly, preferably prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGFxcex2; of TGFxcex1.
In preferred embodiments, the method further includes administering a second treatment) to the transgenic animal. The second treatment can be one which injures or damages the skin, kills skin cells, or can include the removal of hair, e.g., by plucking, shaving, or application of a depilatory, or in general, induces an unwanted condition of the skin. The second treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the treatment can be determined in the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.
In another aspect, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for its effect on skin. The method includes:
providing a transgenic animal, e.g., a mouse, having a reporter gene coupled to a, preferably human, matrix metalloproteinase promoter;
administering the treatment to the transgenic animal, or to a tissue taken therefrom; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the matrix metalloproteinase promoter is a human matrix metalloproteinase promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments, the treatment is administered repeatedly, preferably prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGFxcex2; of TGFxcex1.
In preferred embodiments, the method further includes administering a second treatment to the transgenic animal. The second treatment can be one which injures or damages the skin, kills skin cells, or can include the removal of hair, e.g., by plucking, shaving, or application of a depilatory, or in general, induces an unwanted condition of the skin. The second treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the treatment can be determined in the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.
In another aspect, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for its effect on skin. The method includes:
providing a transgenic animal, e.g. a mouse, having a reporter gene coupled to a vascular endothelial growth factor promoter;
administering the treatment to the transgenic animal; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the vascular endothelial growth factor promoter is a human vascular endothelial growth factor promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments, the treatment is administered repeatedly, preferably prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGFxcex2; of TGFxcex1.
In preferred embodiments, the method further includes administering a second treatment to the transgenic animal. The second treatment can be one which injures or damages the skin, kills skin cells, or can include the removal of hair, e.g., by plucking, shaving, or application of a depilatory, or in general, induces an unwanted condition of the skin. The second treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the treatment can be determined in the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.
In another aspect, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for its effect on skin. The method includes:
providing a transgenic animal, e.g., a mouse, having a reporter gene coupled to a, preferably human, hyaluronan synthase promoter;
administering the treatment to the transgenic animal; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the hyaluronan synthase promoter is a human hyaluronan synthase promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments, the treatment is administered repeatedly, preferably prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGFxcex2; of TGFxcex1.
In preferred embodiments, the method further includes administering a second treatment to the transgenic animal. The second treatment can be one which injures or damages the skin, kills skin cells, or can include the removal of hair, e.g., by plucking, shaving, or application of a depilatory, or in general, induces an unwanted condition of the skin. The second treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the treatment can be determined in the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.
In another aspect, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for its effect on skin. The method includes:
providing a transgenic animal, e.g., a mouse, having a reporter gene coupled to, a preferably human, MMP2 or MMP9, preferably a MMP9, promoter;
administering the treatment to the transgenic animal, or to a tissue taken therefrom; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, a MMP2 or MMP9, preferably a MMP9, promoter is a human MMP2 or MMP9, preferably a MMP9, promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments, the treatment is administered repeatedly, preferably prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGFxcex2; of TGFxcex1.
In preferred embodiments, the method further includes administering a second treatment to the transgenic animal. The second treatment can be one which injures or damages the skin, kills skin cells, or can include the removal of hair, e.g., by plucking, shaving, or application of a depilatory, or in general, induces an unwanted condition of the skin. The second treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the treatment can be determined in the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.
In another aspect, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for its effect on skin. The method includes:
providing a transgenic animal, e.g., a mouse, having a reporter gene coupled to a, preferably human, neutrophil elastase promoter;
administering the treatment to the transgenic animal, or to a tissue taken therefrom; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the neutrophil elastase promoter is a human neutrophil elastase promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments, the treatment is administered repeatedly, preferably prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGFxcex2; of TGFxcex1.
In preferred embodiments, the method further includes administering a treatment (other than the compound) to the transgenic animal. The treatment can be one which injures or damages the skin, kills skin cells, or in general, induces an unwanted condition of the skin. The treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the compound can be determined in the presence of the treatment, and optionally compared to the response seen in the absence of the treatment.
In another aspect, the invention features, a non-human transgenic animal described herein, e.g., a transgenic animal having a reporter gene coupled to a versican promoter.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, see Takeda et al., 1991, L. Am. Geriatr. 39:911-919, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the versican promoter is a human versican promoter.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In another aspect, the invention features, a non-human transgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a matrix metalloproteinase promoter.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the matrix metalloproteinase promoter is a human matrix metalloproteinase promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In another aspect, the invention features, a non-human transgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a vascular endothelial growth factor promoter.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the vascular endothelial growth factor promoter is a human vascular endothelial growth factor promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In another aspect, the invention features, a non-human transgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a hyaluronan synthase promoter.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the hyaluronan synthase promoter is a human hyaluronan synthase promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In another aspect, the invention features, a non-human transgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a Type IV collagenase promoter.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, a MMP2 or MMP9, preferably a MMP9, promoter is a human a MMP2 or MMP9, preferably a MMP9, promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In another aspect, the invention features, a non-human transgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a neutrophil elastase promoter.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In particularly preferred embodiments, the neutrophil elastase promoter is a human neutrophil elastase promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In another aspect, the invention features a promoter-reporter gene construct described herein.
In another aspect, the invention features a method of analyzing GFP presence or distribution in a tissue. The method includes:
providing a tissue sample, e.g., a tissue section, which includes GFP;
evaluating or detecting fluorescent emission, or the lack of fluorescent emission, wherein said detecting step is performed prior to washing or fixing with an aqueous solution,
thereby analyzing GFP in a tissue.
In preferred embodiments the tissue is frozen prior to the detection step. The sample is not contacted with a fixing agent prior to detection.
In preferred embodiments: the tissue is from a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, see Takeda et al., 1991, L. Am. Geriatr. 39:911-919, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging; the GFP is expressed from a transgenic sequence encoding GFP or under the control of a transgenic control element, e.g., a transgenic promoter or enhancer.
In preferred embodiments the promoter is a human promoter.
In preferred embodiments detection includes examination of the sample with a microscope, e.g., a fluorescent or epi-fluorescent microscope.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
Animals described herein can be used in this method.
In another aspect, the invention features, a method of determining the stage of the hair cycle in an animal which expresses a reporter molecule in hair follicle, e.g., the outer root sheath of the hair follicle. The method includes evaluating or detecting the presence or absence of reporter expression, presence being associated with a growing hair cycle (anagen) and absence with a resting hair cycle (telegen and catogen).
In preferred embodiments the reporter molecule is under the control of a promoter expressed in the hair follicle, e.g., the outer root sheath.
In preferred embodiments the promoter is a human promoter.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter molecule is GFP.
In preferred embodiments the animal is a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging;
Animals described herein can be used in this method.
In preferred embodiments the promoter is a VEGF promoter, a versican promoter, or other promoter described herein.
In another aspect, the invention features, a method of analyzing wound healing. The method includes: providing an animal which expresses a reporter molecule under the control of a VEGF promoter, detecting the presence or absence of reporter molecule in a wound, thereby analyzing wound healing.
In preferred embodiments the detection step is reported.
In preferred embodiments, the animal, tissue from the animal, is subjected to a treatment, e.g., the administration of a compound. In such embodiments the method can be used to evaluate the effect of the treatment on wound healing. It may be desirable to compare results from a treated subject or tissue with an untreated subject or tissue.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In preferred embodiments the reporter molecule is GFP.
In preferred embodiments the animal is a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging.
Animals described herein can be used in this method.
In preferred embodiments the promoter is a human promoter.
In another aspect, the invention features, a method of analyzing GFP expression in a transgenic animal having a GFP transgene. The method includes:
a first step of evaluating or detecting the presence or absence of GFP in the animal or in a tissue from the animal; and (optionally)
a second step of evaluating or detecting the presence or absence of GFP in the animal or in a tissue from the animal; and thereby analyzing GFP expression.
In preferred embodiments (in this method and in other methods disclosed herein) the GFP is red shifted GFP.
In preferred embodiments the animal is a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. An animal described herein can be used in this method.
In preferred embodiments at least 1, 5, 10, 20, 30, or 60, 180, 365 days elapse between first and second step.
In preferred embodiments the tissue is skin tissue.
In preferred embodiments the detection steps are performed on a live animal.
In preferred embodiments the promoter is a human promoter.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In another aspect, the invention features, a method of analyzing the expression of a transgene on a transgenic animal, e.g., a transgenic mouse or pig. The method includes:
providing a live transgenic animal;
evaluating or detecting the presence or absence of a reporter gene, e.g., GFP, encoded by a transgenic sequence or under the control of a transgenic control element, e.g., a promoter or enhancer;
thereby analyzing the expression of a transgene.
In preferred embodiments the animal is a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. Animals described herein can be used in the methods.
In preferred embodiments the reporter is under the control of a VEGF promoter or another promoter described herein.
In preferred embodiments the promoter is a human promoter.
In preferred embodiments the promoter is one which is expressed on the skin.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the tissue is skin tissue.
In preferred embodiments the detection steps are performed on a live animal.
In preferred embodiments the method is repeated at least once during the life of the animal. The first and a subsequent repetition of the method can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
In another aspect, the invention features, a method of evaluating gene expression in a live animal. The method includes:
providing a live transgenic animal having a transgene which includes a reporter gene, e.g., GFP, e.g., red shifted GFP;
evaluating or detecting the presence or absence of reporter molecule, e.g., GFP, in the live transgenic animal, thereby evaluating gene expression in the live animal.
In preferred embodiments, the sequence which encodes a reporter gene is under the control of a preselected promoter, e.g., a human promoter. The preselected promoter can be, a skin metabolism-related promoter, e.g.,: a promoter from a gene which encodes a transmembrane protein or a component of the extracellular matrix, such as a proteoglycan promoter, e.g., a versican promoter; a promoter from a protease expressed in the skin, e.g., a matrix metalloproteinase (MMP) promoter, e.g., an MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, or MMP9 promoter; a promoter from a gene which affects vascular function, e.g., a vascular endothelial growth factor promoter; a hyaluronan synthase promoter, e.g., a hyaluronan synthase 1 promoter, a hyaluronan synthase 2 promoter, or a hyaluronan synthase 3 promoter; a promoter for a collagenase expressed in the skin, e.g., a MMP2 or MMP9, preferably a MMP9, promoter; or a neutrophil elastase promoter.
The VEGF promoter is a preferred promoter. In preferred embodiments the promoter is one which is up or down regulated in inflammatory angiogenesis, or neoplastic growth.
In preferred embodiments, the animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In preferred embodiments, a treatment is administered to the animal any of before, during, or after valuation of reporter gene expression. The treatment can be the administration of a compound. The compound can be administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.
In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.
Methods of the invention can be performed in vivo, with whole animals, or in vitro, that is, with tissue, e.g., skin, or cells, which are derived from a transgenic animal described herein or with cells, preferably skin cells or tissue, from cells transformed with a skin-metabolism promoter/reporter gene construct.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is an animal, e.g., a non-human mammal, e.g., a mini-pig, a guinea-pig, or a rodent, e.g., a mouse or a rat, in which one or more, and preferably essentially all, of the cells of the animal include a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, e.g., by microinjection, transfection or infection, e.g., by infection with a recombinant virus. The term genetic manipulation is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
Transgenic animals can be, e.g., heterozygous or homozygous for a transgene.
As used herein, the term xe2x80x9crodentxe2x80x9d refers to all members of the phylogenetic order Rodentia.
As used herein, the term xe2x80x9creporter genexe2x80x9d refers to a nucleic acid sequence which is fused downstream of a skin metabolism-related promoter, such that its expression is under the control of the promoter. Reporter genes usually encode a protein whose activity can be easily measured. For example, the reporter gene can be a gene encoding an assayable protein, e.g., an enzyme, not found in the cell in nature, e.g., a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, a chloramphenicol acetyl transferase gene, luciferase, and the like. In a preferred embodiment the reporter product is capable of providing a signal which indicates the activity of the promoter to which it is linked. Preferred reporters are those which luminesce or fluoresce. Preferred reporters can luminesce or fluoresce, in vivo, without the addition of an exogenous substrate. A particularly suitable reporter is green fluorescent protein. Modified variants of green fluorescent protein, e.g., EGFP, EBFP, EYFP, d2EGFP, ECFP, GFPuv are included within the term green fluorescent protein. EGFP is particularly preferred. These variants of GFP are commercially available by C1ONTECH, Laboratories, Inc. Palo Alto, Calif. Furthermore, GFP and variants thereof, are provided in the following references, all of which are incorporated by referenced: Chalfie, M. et al. (1994) Science 263:802-805; Prasher, D. C., et al. (1992) Gene 111:229-233; Inouye, S. and Tsuji, F. I. (1994) FEBS Letters 341:277-280; Wang, S. and Hazelrigg, T. (1994) Nature 369:400-403; Cody, C. W., et al. (1993) Biochemistry 32:1212-1218; Inouye, S. and Tsuji, F. I. (1994) FEBS Letters 351:211-214; Heim, R., et al. (1994) Proc. Natl. Acad. Sci., USA 91:12501-12504; Yang, T. T., et al. (1996) Nucleic Acids Res. 24(22): 4592-4593; Cormack, B. P., et al. (1996) Gene 173:33-38; Crameri, A., et al. (1996) Nature Biotechnol. 12:315-319; Haas, J. et al, (1996) Curr. Biol. 6:315-324; Galbraith, D. W., et al. (1995) Methods Cell Biol. 50:1-12; Living Colors Destabilized EGFP Vectors (April 1998) CLONTECHniques XIII(2):16-17, Living Colors pEBFP Vector (April 1997) CLONTECHniques XII(2):16-17; Heim, R. and Tsien, R. Y. (1996) Curr. Biol. 6:178-182;
Ormxc3x6, et al. (1996) Science 273:1392-1395; Mitra, R. D. et al. (1996) Gene 173:13-17.
As used herein, the term xe2x80x9cskin metabolism-related promoterxe2x80x9d refers to a promoter which is transcriptionally active in the skin. It need not be skin-specific. The gene in which the promoter is naturally found can be a gene involved in the maintenance, or proper functioning of the skin. For example, the gene can be a gene encoding a protein which is part of the extracellular matrix, a protein involved in the maintenance or degradation of the extracellular matrix, or a protein involved in supplying nutrition to the skin. Active fragments or analogs of the promoters mentioned herein can be used in methods, compositions, and animals of the invention, e.g. an active fragment of the veriscan promoter. The 839 bp fragment of the functional human veriscan promoter (xe2x88x92559 to +280) (Naso, M. F. , Zimmermann, D. R. and Iozzo, R. V. (1994) J. Biol. Chem., 269, 32999-33008) human genomic DNA can be used in methods which use a veriscan promoter.
Embodiments of the inventions allow for evaluation of transgene expression, in situ, on live animals. The luminescent or fluorescent reporters, e.g., GFP reporter molecule, are particularly advantageous in such embodiments.
As used herein, xe2x80x9cadministering a compound to an animalxe2x80x9d refers to dispensing, delivering or applying a treatment to an animal or cell. Administration can be by topical administration, by parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery or administration by the intranasal or respiratory tract route. The most preferred administrations are topical application or subcutaneous or intradermal injection.
The methods of the invention allow rapid and efficient evaluation of compounds for their effect on skin.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.